人重组IL-17蛋白的表达及其生物学特性的初步分析

发布时间：2018-02-01 07:17

本文关键词： IL-17 Th17 融合蛋白包涵体复性 HeLa　出处：《苏州大学》2007年硕士论文　论文类型：学位论文

【摘要】： Th1-Th2的经典分类的提出已经有20年了，它为我们深入理解固有免疫和适应性免疫之间的相互联系提供了新的思路和研究模型。然而随着IL-17和IL-12两个家族的细胞因子的发现和研究，这种分类方法逐渐被予以改变：认为机体内存在以分泌IL-17为特征的T细胞亚群，和以往的Th1，Th2都不同，这群细胞在IL-23，IL-6和IL-1诱导后产生IL-17细胞因子，并发挥独特的生物学效应，从而解释了以前在免疫调节，宿主抵抗病菌，免疫病理等方面未能解释的问题。这种T细胞现在命名为Th17细胞。它的特异性在于能分泌IL-17细胞因子，参与体内多种免疫反应。Th17细胞数量和IL-17在机体内表达水平的改变，以及Th17细胞和Th1细胞比例的变化与疾病的发展密切相关并成为广为关注的研究课题。为此，本研究旨在表达重组蛋白和生物学特性分析作为切入点，为本单位开展相关工作奠定了一定的物质基础。一．人IL-17基因全长及去信号肽片段的克隆和载体的构建根据文献报道的人IL-47的基因序列设计合成了全长和去信号肽的特异性引物。采用RT-PCR方法从人外周血PHA刺激活化的T细胞中扩增了人IL-17全长基因和去信号肽基因。应用双酶切法酶切载体和目的基因，再用连接酶连接酶切回收后的产物。测序正确后，在TOP10宿主菌中增殖PQE3.0／IL-17和PCEP4／IL-17质粒，经酶切和PCR鉴定后PQE3.0／IL-17转化M15表达菌株。二．人PQE3.0／IL-17融合蛋白在大肠杆菌中的表达和鉴定在LB细菌培养基中增殖M15表达菌，采取一系列实验条件，包括降低温度，缩短诱导时间等，均证实不能诱导可溶性IL-17融合蛋白的表达，而表达的蛋白主要集中在包涵体内。表达动力学分析表明，应用1 mmol／L IPTG诱导5 h可获得最有效的表达。实验室规模增殖表达菌，经超声裂解和离心，沉淀变性及复性，透析后经HiTrap亲和层析柱一步纯化融合蛋白。实验结果表明，纯化后的IL-17／His融合蛋白纯度可达90％以上，定量为2 mg／ml。应用Western-blot对融合蛋白进行生物学鉴定，结果为约15KD的单一条带。三．人IL-17在真核系统中的表达抽取PCEP4／IL-17质粒，转染CHO细胞，应用潮霉素进行筛选，在显微镜下观察到阳性克隆，胰蛋白酶消化细胞，再用移液尖挑取阳性克隆分孔培养，继续使用药物筛选，直到所获得的克隆表达稳定为止。吸取阳性克隆细胞培养的上清，用ELISA方法定性和定量检测IL-17的表达情况。实验的结果是所获得表达量较高的融合蛋白。四．人IL-17重组蛋白的生物学功能的初步研究采用人的宫颈癌细胞株HeLa作为反应细胞，按不同浓度加入IL-17／His融合蛋白和IL-17／Fc融合蛋白，反应48小时后，用ELISA法检测上清IL-6和GM-CSF的水平，与对照组作比较，观察两种IL-17蛋白对HeLa细胞的作用在一定剂量范围内是否存在剂量依赖关系以及两种蛋白活性度的高低。综上所述，本项研究我们成功地应用原核和真核系统表达出了人IL-17重组蛋白，并进行了生物学活性的初步研究。所表达的蛋白具有一定的生物学活性，为我们下一步工作奠定了基础。涉及IL-17锚定蛋白和单克隆抗体的研制工作尚待下一步继续展开。
[Abstract]:The classic Th1-Th2 classification has been proposed for 20 years, which provides new ideas and research model for us to further understand the relationship between innate and adaptive immunity. However with cytokines IL-17 and IL-12 in two families, the discovery and research of this classification method has been changed to that body to the secretion of IL-17 of T cell subsets, and the previous Th1, Th2 are different, this group of cells in IL-23, IL-17, IL-6 and IL-1 induced cytokine production, and play biological effects unique, thus explaining the former in immune regulation, immune host defense against pathogen, pathology and other aspects of the problem. This can not explain T cells now named Th17 cells. Its specificity is IL-17 can secrete cytokines, participate in the change of expression level and the number of IL-17.Th17 cells in vivo immune response in the body, and Th17 The change of cell to Th1 cell ratio is closely related to the development of disease and has become a research topic of widespread concern. Therefore, the purpose of this study is to express recombinant protein and biological characteristics as a breakthrough point, laying a solid foundation for the related work of our unit.
The cloning and construction of the full length of human IL-17 gene and the fragment of the de signal peptide
According to the length and specific primers were designed and synthesized to signal peptide gene sequences reported in the literature. IL-47 from human peripheral blood PHA stimulated by RT-PCR method in T cells amplified human IL-17 full-length gene and signal peptide gene. The application of double enzyme digestion enzyme carrier and target genes, and ligase the digestion products recovered. Ligase after sequencing, the TOP10 host cell proliferation of PQE3.0 / IL-17 and PCEP4 / IL-17 plasmids were identified by enzyme digestion and PCR after PQE3.0 / IL-17 conversion M15 expression strain.
Two. Expression and identification of human PQE3.0 / IL-17 fusion protein in Escherichia coli
The expression of M15 in bacteria proliferation medium LB bacterial culture, take a series of experimental conditions, including lowering the temperature, shorten the induction time, confirmed the expression of soluble IL-17 fusion protein could not be induced, and the expression of the protein is mainly concentrated in the inclusion body. The expression kinetics analysis shows that the application of 1 mmol / L 5 h induced IPTG expression can be obtained the most effective laboratory scale. The proliferation of expression of bacteria, after ultrasonic lysis and centrifugation, precipitation denaturation and renaturation after dialysis by HiTrap affinity chromatography purified fusion protein. The experimental results show that the purified IL-17 / His fusion protein reached a purity of more than 90%, 2 mg / ml. for the quantitative application of Western-blot fusion protein the biological identification results for a single band of about 15KD.
Three. Expression of human IL-17 in the eukaryotic system
Extraction of PCEP4 / IL-17 plasmid was transfected into CHO cells using hygromycin screening, positive clones were observed under the microscope, trypsin digestion and pipette tip cells, positive clones with hole culture, continue to use drug screening, cloning and expression of the stable until now. Pipeted the cell culture supernatant, with the expression of ELISA method for qualitative and quantitative detection of IL-17. The experimental results are obtained by the high expression of fusion protein.
Four. Preliminary study on the biological function of human IL-17 recombinant protein
The human cervical cancer cell line HeLa as the reaction cells with different concentration adding IL-17 / His fusion protein and IL-17 / Fc fusion protein, 48 hours after the reaction, ELISA was used to detect the supernatant of IL-6 and GM-CSF levels, compared with the control group, to observe the effect of two kinds of IL-17 protein in HeLa cells in a certain dose in the range of dose dependent and two kinds of high and low protein activity.
In summary, in this study we successfully used prokaryotic and eukaryotic expression system. Recombinant human IL-17 protein, and studied the biological activity. The expression protein has certain biological activity, for our next work laid the foundation for development work. Relates to IL-17 anchored protein and monoclonal antibody is to be the next step to continue to expand.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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